Carotenoids are yellow, red and orange pigments and are widely distributed in nature. Although specific carotenoids have been identified in various fruits and vegetables, bird feathers, egg-yolk, poultry skin, crustaceans and macular eye region, they are especially abundant in marigold petals, corn and leafy vegetables. The correlation between dietary carotenoids and carotenoids found in human serum and plasma indicate that only selected groups of carotenoids make their entry into the human blood stream to exert their effect. Each carotenoid shows an individual pattern of absorption, plasma transport and metabolism.
Carotenoids absorb light in the 400-500 nm region of the visible spectrum. This physical property imparts the characteristic yellow/red color to the pigments. Carotenoids contain a conjugated backbone composed of isoprene units, which are usually inverted at the center of the molecule, imparting symmetry. Changes in geometrical configuration about the double bonds result in the existence of many cis- and trans-isomers. Mammalian species do not synthesize carotenoids and therefore these have to be obtained from dietary sources such as fruits, vegetables and egg yolks. In the recent years, carotenoids have been reported to have several health benefits, which include prevention and/or protection against serious health disorders.
Carotenoids are non-polar compounds classified into two sub-classes, namely, polar compounds called xanthophylls or oxy-carotenoids and non-polar hydrocarbon carotenes like β-carotene, lycopene, etc. Both the sub-classes have at least nine conjugated double bonds responsible for the characteristic colors of the carotenoids. Xanthophylls have ring structures at the end of the conjugated double bond chain with polar functions like hydroxyl or keto group. The examples for xanthophylls include lutein, zeaxanthin, capsanthin, canthaxanthin, β-cryptoxanthin, astaxanthin, etc. As natural colorants and also for their role in human health, xanthophylls like lutein and zeaxanthin have attracted the renewed attention of scientists and researchers in the biomedical, chemical and nutritional field in recent years.
Lutein and zeaxanthin contribute to yellow and orange—yellow colours respectively. Lutein and zeaxanthin can be present in plant material in the free form and also in ester form. Lutein is present in green leafy vegetables like spinach, kale and broccoli in the free form; fruits like mango, orange and papaya; red paprika, algae, yellow corn, contain lutein in the form of its esters. It is also present in the blood stream and various tissues in human body and particularly in the macula, lens and retina of the eye.
Lutein is chemically designated as β,ε-carotene 3,3′-diol. Zeaxanthin is formed by the addition of two hydroxy groups to β-carotene. Since the hydroxy positions are in 3 and 3′-, the chemical name for zeaxanthin is β,β-carotene-3,3′-diol. The common name of zeaxanthin is derived from zea mays because this carotenoid was first identified in corn (zea mays).
It can be seen that lutein is not symmetrical as the position of double bond in the left ring is not identical with the double bond position in the right ring. Zeaxanthin is completely symmetrical with regards to left and right rings due to an extra conjugated double bond compared to lutein.
Xanthophylls can show both optical (R- and S-stereo isomers) and geometrical isomers (trans,E- and cis,Z-). The conformation of R- and S-stereo isomers is based on CD spectral and chiral column HPLC studies while the conformation of cis- and trans-isomers is based on electronic, infrared, NMR, HPLC-MS and HPLC-NMR on-line spectroscopy studies. It is well known that when an organic molecule has a carbon atom with four different types of atoms or groups attached to it, that carbon atom is designated as chiral carbon atom. The chiral carbon atom is responsible for two different spatial arrangements leading to formation of optical isomers while the number of double bonds of the polyene chain and the presence of a methyl group and the absence of steric hindrance decide the number of trans- and cis-isomers. In the case of trans-zeaxanthin, the carbon atoms at 3 and 3′ positions in the two end rings are both chiral atoms. Thus, trans-zeaxanthin has two chiral centers at the carbon atoms C3 and C3′, based on the positions of the secondary hydroxy groups attached to them. Therefore, there are four possible stereo isomers of trans-zeaxanthin namely, (3R-3′R)-isomer, (3S-3′S)-isomer and (3R-3′S)- or (3S-3′R)-isomer. In these isomers (3R-3′S)- & (3S-3′R) are identical. Thus, there are three chiral isomers of trans-zeaxanthin. The isomer causing rotation of polarized light in a right handed manner is called R-stereo isomer, the isomer causing left handed rotation S-stereo isomer and the third isomer possessing a two fold opposite effects (optically inactive) which is called meso-form of trans-zeaxanthin. These are shown in the formulae given below including the chemical structure of lutein

The conjugated double bonds of lutein and zeaxanthin contribute to the distinctive colours of each pigment, and also influence the ability of these to quench singlet oxygen. Due to the extra conjugated double bond, zeaxanthin is believed to be a stronger anti-oxidant compared to lutein.
The macular pigment of the eye is composed primarily of three xanthophylls pigments, namely (R,R)-lutein, (R,R)-zeaxanthin and (R,S)-zeaxanthin in the order 36, 18 and 18% of the total carotenoid content of the retina along with the remaining 20% consisting of minor carotenoids like oxo-lutein, epi-lutein and ε,ε-carotene 3,3′-dione (J. T. Landrum and R. A. Bone, Arch. Biochem Biophys, 385, 28(2001)). Although these xanthophyll pigments are found throughout the tissues of the eye, the highest concentration is seen in the macula lutea region of the retina, including a central depression in the retina called the fovea. The concentration of xanthophylls pigments increases progressively towards the center of the macula and in the fovea, the concentration of these xanthophyll pigments are approximately a thousand fold higher than in other human tissues. (Landrum et al., Analysis of zeaxanthin distribution within individual human retinas, Methods in Enzymology, L. Packer (editor) 213A, 457-467, Academic Press 1992). The fovea is a relatively small area within the macula, in which the cone photoreceptors reach their maximal concentration. About 50% of the total amounts of the xanthophylls is concentrated in the macula where zeaxanthin dominates over lutein by a ratio of 2:1 (Handelman et al., Measurements of carotenoids in human and monkey retinas, in Methods in Enzymology, L. Packer (editor) 213A, 220-230, Academic press, NY, 1992; Billsten et al., Photochemistry and photobiology, 78, 138-145, 2003). At the center of the retinal fovea, zeaxanthin is a 50:50 mixture of (trans-3R,3′R)-zeaxanthin and (trans-3R,3′S)-zeaxanthin along with small quantity of (3S,3′S)-zeaxanthin (J. T. Landrum and R. A. Bone, Arch. Biochem. Biophy., 385, 28 2001).
The fovea is particularly important for proper visual function (e.g., acuity) and disease and damage to this area is known to result in legal blindness. For example, age-related macular degeneration (AMD) is characterized by pathological changes in the retina, retinal pigment epithelium (RPE) and/or the choroid and preferentially affects the macular region of the retina. This is the leading cause of irreversible vision loss in the United States among those ≧65 y old and there is no established treatment available for most patients. The loss of central vision results in the possible inability to recognize faces, to read or drive a car and therefore has a significant effect on an individual's ability to live independently. There is ample epidemiologic evidence which supports a role for dietary intake of lutein and zeaxanthin in different isomeric forms in protection against age-related cataract and macular degeneration. The detection of oxidation products of lutein and zeaxanthin in the human retina supports the hypothesis that dietary lutein and zeaxanthin may act as antioxidants in the macular region. (Khachik et al., Invest. Opthalmol. and Vis. Sci., 38, 1802-1811, 1997)
Of the 40 to 50 carotenoids typically consumed in the human diet, lutein and zeaxanthin, are deposited at an up to 5 fold higher content in the macular region of the retina as compared to the peripheral retina. Zeaxanthin is preferentially accumulated in the foveal region, whereas lutein is abundant in the perifoveal region.
Regarding the location of xanthophylls at a cellular level, they are reported to be bound to specific proteins, xanthophylls binding protein (XBP). The XBP is suggested to be involved in the uptake of lutein and zeaxanthin from the blood stream and stabilization of the same in the retina. The study of xanthophylls and XBP by femto-second transient absorption spectroscopy showed better stability for (3R,3′S)-zeaxanthin enriched XBP compared to (3R,3′R)-zeaxanthin while the photophysical properties of the xanthophylls(3R,3′R)-zeaxanthin and (3R, 3′S,meso)-zeaxanthin are generally identical. It is likely that the meso-zeaxanthin is better accommodated with XBP wherein the protein protects the xanthophylls from degradation by free radicals. Thus, the complex may be a better antioxidant than the free xanthophylls, facilitating improved protection of ocular tissue from oxidative damages. (Billsten et al. Photochemistry and Photobiology, 78, 138-145, 2003)
Several functions have been attributed to macular pigments including the reduction of the damaging effects of photo-oxidation from blue light absorbed by the eye, reduction of the effects of light scatter and chromatic aberration on visual performance, and protection against the adverse effects of photochemical reactions because of the antioxidant properties of the carotenoids.
The ability to increase the amount of maculir pigment by dietary supplementation with lutein has been demonstrated (Landrum et al., Dietary Lutein supplementation increases macular pigment, FASEB. J, 10, A242, 1996). The reduced vision function due to cataract and the adult blindness due to AMD can be substantially controlled by consuming fruits and vegetables and dietary supplements containing lutein and zeaxanthin and meso-zeaxanthin available from sea foods denying the vegetarian population. Although meso-zeaxanthin present in eye is considered a metabolic product originating from lutein, the need for dietary supplementation of meso-zeaxanthin is now recognized to improve the macular pigment density.    (Landrum and Bone, Functional Foods and Nutraceuticals, 10 Sep. 2001). Similarly, the study has shown that R,R-zeaxanthin gains entry to blood and finally to macula.    (Breithaupt et al., Brit. J. Nutr. 91, 707-713, 2004). Lutein and zeaxanthin dietary supplements in human trials have been shown to raise the macular pigment density and serum concentrations of these carotenoids (Bone et al., J. Nutr., 133, 992-998, 2003).Dietary Sources of Lutein and Zeaxanthin
Lutein is a common carotenoid found in most fruits and vegetables, while zeaxanthin in the (R,R)-isomer form is present only in minute quantities in most fruits and vegetables. Dietary sources of zeaxanthin are limited to greens, certain yellow/orange fruits and vegetables such as corn, nectarines, oranges, papaya, persimmons and squash. Capsicum annum is another most common spice widely used which is a good source of zeaxanthin. Wolfberry (Lycium barbarum, fructus lycii or Gou Qi Zi) plant has small red berries which are commonly used in Chinese home cooking and has been shown to have a high content of zeaxanthin (mainly as zeaxanthin dipalmitate) but negligible amounts of lutein. The dried fruit of wolfberry is prescribed by Chinese herbalists as a therapeutic agent for a number of eye diseases. In France, lutein dipalmitate (Helenien) isolated from the blossom leafs of Helenium autumnale is reported to be used for the treatment of the visual disorders.
As already mentioned earlier, the dietary source of meso-zeaxanthin is mainly from seafoods like shrimps, fish, turtle, etc, thereby the vegetarian population is deprived of meso-zeaxanthin. However, there are patents available for pharmaceutical compositions containing meso-zeaxanthin for treatment of retinal disorders like increasing the deposition of macular pigments in the human eye and therapeutic treatment or prophylaxis of AMD (Howard et al., U.S. Pat. No. 6,329,432, 2001).
Lutein and zeaxanthin occur naturally in trans-isomeric form in fruits, vegetables and flowers (marigold). Because of processing conditions due to heat and light, a small percentage of trans- is converted into cis-isomeric form. Therefore, the preferred bio-available form is trans-isomeric as evidenced from the data of geometric isomers compositional analysis of human plasma. (Khachik et al., Isolation and structure elucidation of geometric isomers of lutein, zeaxanthin in extracts of human plasma, J. Chrom. 582, 153-156, 1992). In view of this, it is desirable to use the trans-isomeric form of meso-zeaxanthin in dietary supplements.
To date little is known about the mechanism of formation, uptake and deposition of meso-zeaxanthin in the retina of the eye. Khachik et al. have reported the presence of 2-3% of (3R,3′S, meso)-zeaxanthin in twenty normal human plasma samples and proposed the metabolic pathways of its formation from dietary lutein and zeaxanthin. It is not clear whether the deposition of meso-zeaxanthin in the retina routes through serum or are produced from lutein/zeaxanthin within the retina. (Khachik. et al., in a chapter on Dietary carotenoids and their metabolites as potentially useful chemoprotective agents against cancer, in “Antioxidant food supplement in human health, Eds. Packer et al., Academic Press London, page pp 203-29, 1999). However, Breithaupt et al. did not find the presence of meso-zeaxanthin in human plasma obtained 24 hrs after ingestion of (3R,3′R)-zeaxanthin (ester or free form) in a single blind cross over study using two groups each consisting of six volunteers. The chiral LC-ApcI-MS was used for detection in the pooled plasma sample. (Brit. J. Nutri. 91, 707, 2004)
There is evidence and reasons supporting the hypothesis that the carotenoids lutein, zeaxanthin and meso-zeaxanthin are readily bio-available and consequently increase macular pigment levels (Landrum and Bone, Meso-zeaxanthin-A cutting edge carotenoid, Functional Foods and Nutraceuticals, 10 Sep. 2001).
In present days, there is high demand for xanthophyll crystals containing high amounts of trans-lutein and/or zeaxanthin for its use as antioxidants, prevention of cataract and macular degeneration, as lung cancer-preventive agent, as agents for the absorption of harmful ultra-violet light from sun rays and quencher of photo-induced free radical and reactive oxygen species, etc. A number of commercial products from natural source are now available to facilitate the formulation of industrial and commercial products with lutein or (R,R)-zeaxanthin. However, high concentration (trans, 3R,3′S,meso)-zeaxanthin concentrates, or standardized products for industrial application derived from the same natural source as commercial lutein or zeaxanthin are still not widely available.
As trans-xanthophylls occur naturally in foods with good stability and in greater bio-vailability compared to corresponding cis-isomers, it would be useful for industry and nutritional product formulators to have (trans,3R,3′S,meso)-zeaxanthin from a commercial scale process, made from natural source material same as that which is already accepted by the market for lutein and zeaxanthin products, namely marigold. Unlike products made from synthesis routes, such a trans, meso-zeaxanthin product should be made from safe solvents (GRAS solvents) for producing dietary supplements suitable for human consumption, with minimal solvent residues and specific ratios of lutein and zeaxanthin isomers keeping in mind market requirements.